金丝桃苷对地塞米松诱导骨髓间充质干细胞骨分化的影响

Effect of hyperoside on dexamethasone-induced osteogenic differentiation of bone marrow mesenchymal stem cells

摘要:

[目的]观察金丝桃苷（hyperoside）对地塞米松（dexamethasone, Dex）诱导的小鼠骨髓间充质干细胞（bone marrow mesenchyml stem cells, BMSCs） 增殖和成骨分化抑制的影响及分子机制。[方法] 小鼠 BMSCs，分为空白对照组 （blank control, BC）、地塞米松组 （Dex 组）、Dex+ Hyperoside 1 μmol/L 组 （DH-1）、Dex+ Hyperoside 2 μmol/L 组 （DH-2）、Dex+ Hyperoside 5 μmol/L 组（DH-5）。采用 CCK8 法检测细胞活力，诱导 BMSCs 成骨分化后，检测碱性磷酸酶（Alkaline phosphatase, ALP）和茜素红染色细胞矿化；qRT-PCR 法检测 ALP、Runx2 和 Osterix mRNA 相对表达量；Western blot 法检测 PI3K、p-PI3K、AKT、pAKT 蛋白表达量。[结果]BMSCs 细胞活性由高至低依次为：BC 组＞金丝桃苷各浓度组（DH-1 组、DH-2 组、DH-5 组）＞ Dex 组，差异有统计学意义（P＜0.05）；DH-1 组、DH-2 组和 DH-5 组细胞活性两两比较差异无统计学意义（P＞0.05）。ALP 活性由高至低依次为：BC 组＞DH-5＞DH-2＞DH-1＞Dex 组，整体差异有统计学意义（P＜0.05）。成骨诱导 14 d 后，Dex 组矿化结节形成最少，随着 Hyperoside 浓度的增加，矿化结节数量逐渐增多，呈现一定的剂量依赖性。ALP、Runx2 和 Osterix mRNA 表达水平由高至低均依次为 BC 组＞Hyperoside 各浓度组（DH-5 组、DH-2 组、DH-1 组）＞Dex 组，整体差异有统计学意义（P＜0.05）。与 BC 组相比，Dex 组 p-PI3K/PI3K [(1.0±0.2) vs (0.4±0.1), P＜0.05] 和 p-AKT/AKT [(1.0±0.1) vs (0.6±0.1), P＜0.05]比值均显著降低。与 Dex 组相比，Hyperoside 组 p-PI3K/PI3K [(0.4±0.1) vs (0.8±0.1), P＜0.05] 和 p-AKT/AKT [(0.6±0.1) vs (0.9±0.1) , P＜0.05] 比值显著升高 （P＜0.05）。[结论]Hyperoside 通过 PI3K/AKT 信号通路减轻了 Dex 诱导的 BMSCs 增殖和成骨分化抑制。

Abstract:

[Objective] To explore the effect of hyperoside on dexamethasone (Dex)-induced inhibition of proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and the potential molecular mechanism. [Methods] The BMSCs were isolat- ed from mice and cultured in vitro, and were divided into blank control group (BC), Dex group, Dex + hyperoside 1 μM group (DH-1), Dex + hyperoside 2 μM group (DH-2) and Dex + hyperoside 5 μM group (DH-5). After the osteogenic differentiation of BMSCs was induced, the cell viability was measured by CCK8 assay, the activity of alkaline phosphatase (ALP) was detected using ALP activity kit, and the mineral- ization level was observed by Alizarin Red S staining. In addition, the mRNA expressions of ALP, Runx2, and Osterix were measured by qRT-PCR, while protein expressions of PI3K, p-PI3K, AKT, and p-AKT in BMSCs were determine by western blot assay. [Results] The cell vitality of BMSCs was ranked from high to low as follows: BC group ＞ every hyperoside groups (DH- 1 group, DH- 2 group, DH- 5 group) ＞ Dex group, which was statistically significant (P＜0.05), despite that there was no significant difference in cell vitality among DH-1 group, DH-2 group and DH-5 group (P＞0.05). The ALP activity was up-down as BC group ＞DH-5＞DH-2＞DH-1＞Dex, with a statistically significant overall difference (P＜0.05). After 14 days of osteogenic induction, the formation of mineralized nodules in Dex group was the least, while which gradually increased with the increase of Hyperoside concentration in a certain dose dependence manner. The mRNA ex- pressions of ALP, Runx2 and Osterix were ranked up-down as the BC＞DH-5＞DH-2＞DH-1＞Dex, with a statistically significant overall dif- ference among groups (P＜0.05). Compared with those in the BC group, the p-PI3K/PI3K ratio [(1.0±0.2) vs (0.4±0.1), P＜0.05] and p-AKT/ AKT ratio [(1.0±0.1) vs (0.6±0.1), P＜0.05] in the Dex group significantly reduced. However, compared with those in Dex group, the p-PI3K/ PI3K ratio [(0.4±0.1) vs (0.8±0.1), P＜0.05] and p-AKT/AKT [(0.6±0.1) vs (0.9± 0.1), P＜0.05] in the hyperoside group significantly in-creased. [Conclusion] The hyperoside might alleviates Dex- induced inhibition of proliferation and osteogenic differentiation of BMSCs through PI3K/AKT signaling pathway.